How should arginine and glucagon stimulation tests be performed to assess growth hormone deficiency in an overnight-fasted child without contraindicating conditions or interfering medications?

How to Perform Arginine and Glucagon Stimulation Tests for Growth Hormone Deficiency in Children

For arginine stimulation testing in children, administer 0.5 g/kg arginine hydrochloride (maximum 30 g) intravenously over 30 minutes after an overnight fast, with blood sampling at -30,0,30,60,90,120, and 150 minutes; for glucagon testing, administer 1 mg intramuscularly (1.5 mg if weight >90 kg) after overnight fast with blood sampling at 0,30,60,90,120,150,180,210, and 240 minutes. 1, 2

Pre-Test Preparation

Both tests require standardized conditions to ensure accurate results:

• Schedule the test in the morning following a normal night's sleep
• Maintain overnight fast through the entire test period
• Ensure bed rest for at least 30 minutes before infusion begins
• Minimize patient apprehension and distress, particularly critical in children
• Verify adequate replacement of other deficient hormones before testing 1

Arginine Stimulation Test Protocol

Dosing

• Pediatric dose: 0.5 g/kg arginine hydrochloride IV over 30 minutes
• Maximum dose: 30 g (regardless of weight)
• For children ≤59 kg: withdraw weight-based dose into separate container to avoid inadvertent overdose
• For children ≥60 kg: use full 300 mL bottle (30 g) 1

Administration Details

• Use indwelling needle or soft catheter in antecubital vein (arginine is hypertonic)
• Draw blood samples from contralateral arm
• Infuse at uniform rate over exactly 30 minutes starting at time zero 1

Sampling Schedule

Blood samples at: -30,0,30,60,90,120, and 150 minutes 1

Important caveat: Recent research suggests restricting to fewer time points increases false-positive rates—sampling at only 60,90,120 minutes on arginine testing showed 8.5% false-positive rate 3. The full 7-point sampling schedule is recommended for accuracy.

Sample Handling

• Promptly centrifuge blood samples
• Store plasma at -20°C until assayed
• Use published radioimmunoassay procedures 1

Glucagon Stimulation Test Protocol

Dosing

• Standard dose: 1 mg intramuscular
• If weight >90 kg: 1.5 mg intramuscular 2

Sampling Schedule

Blood samples at: 0,30,60,90,120,150,180,210, and 240 minutes 2

Critical timing consideration: GH release with glucagon is delayed compared to other secretagogues—monitoring over at least 3 hours is mandatory. Peak GH typically occurs at 120 minutes 2, 4, 3. Restricting to 90,120,150 minutes showed only 3.8% false-positive rate, but full sampling is preferred 3.

Safety Monitoring

• Monitor glucose levels throughout test—glucagon may cause secondary insulin release leading to delayed hypoglycemia
• Check for hypoglycemia even after test completion 5

Interpretation of Results

GH Peak Cut-offs

The interpretation varies by BMI and should account for obesity's blunting effect:

• Standard pediatric cut-off: Peak GH <7-10 μg/L suggests GHD (most commonly 7.5 μg/L used) 6
• Adult cut-offs:
  • Glucagon: >3 μg/L normal (but >1 μg/L in obese individuals) 2, 6
  • General: 3-5 μg/L range depending on assay 6

Important nuance: Data suggest clinicians often misdiagnose GHD using peak values of 5-10 μg/L, with poor test reproducibility being a significant issue 6. The 2018 Endocrine Society guideline emphasizes using the same cut-offs in childhood cancer survivors as the general population 6.

Test Sequence Considerations

Recent evidence strongly favors glucagon as the first test: A 2026 study of 881 children showed glucagon-first had significantly lower discordant results (37.4%) compared to arginine-first (50.7%, p=0.048) 7. Similarly, a 2020 study found clonidine-first more efficient than glucagon-first, with glucagon showing 69.6% divergent results versus 46.4% for clonidine 8.

Practical implication: Consider using glucagon or clonidine as the initial test, reserving arginine for confirmation when the first test shows GH peak between threshold values.

Confirmatory Testing

• If initial test shows deficiency, confirm with second test using different stimulus
• Wait one day between tests 1
• False-positive rate for arginine: approximately 32%
• False-negative rate for arginine: approximately 27% 1

Exception to two-test rule: Testing not required if three other confirmed anterior pituitary hormone deficits are present 6

Special Considerations

Factors That Blunt GH Response (False-Positives)

• Obesity (use lower cut-offs)
• Sex steroid deficiency (consider priming in prepubertal children >11 years)
• Hypothyroidism (must be corrected before testing)
• BMI effects are significant—cut-offs should be BMI-correlated 2, 6

Tests to Avoid in Specific Contexts

Do NOT use GHRH-arginine or GHRH alone in children with hypothalamic damage (e.g., after cranial radiation) as these give falsely normal results 6. Both insulin tolerance test (ITT) and glucagon properly evaluate the complete hypothalamic-somatotroph axis 6.

Assay Variability

Significant discrepancies exist between GH assays using different antibodies and reference standards. The Consensus Statement recommends harmonized assays using IS 98/574 standard, with some experts suggesting a 7 ng/mL cut-off when comparing different immunoassays 9. However, 10 ng/mL remains standard of care in the United States 9.

Age-Related Considerations

Younger children show higher GH responses to glucagon—the magnitude of GH peak after glucagon correlates negatively with age at diagnosis (rho = -0.636, p<0.0001) 4. This suggests age-specific normative data may be needed, though not yet established for routine clinical use.